Foraging for selenium: a comparison between hyperaccumulator and non-accumulator plant species

Selenium (Se) hyperaccumulators are a unique group of plants that can accumulate this element in their aerial parts at concentrations exceeding 100 mg kgDW−1. These plants actively search for Se in the soil, a phenomenon known as root foraging, reported to date only by few studies. In this study, the effect of localized Se enrichment, in the form of selenite and selenate, was investigated on the root architecture of two Se-hyperaccumulators (Stanleya pinnata and Astragalus bisulcatus) and two non-accumulators (Brassica juncea and Medicago sativa). Rhizoboxes were divided into two halves: one half was filled with control soil while the other with selenate or selenite (30 mg kgDW−1) spiked soil. Seedling were transferred into the interface of the two soils and allowed to grow for three weeks under controlled light and temperature conditions. Staneya pinnata exhibited equal root density in both halves of the rhizobox when grown in control/control and selenite/control soil treatments. However, in the presence of selenate, S. pinnata developed 76% of the roots towards the selenate-enriched half, indicating an active root foraging. In contrast, A. bisulcatus and the non-accumulators B. juncea and M. sativa did not show any preferential distribution of roots. This study revealed that only S. pinnata showed the ability to detect and forage for Se when provided as selenate. Non-accumulators did not show any morphological or Se-accumulation difference associated with the presence of Se in soil in either form.


Materials and methods
Rhizobox preparation. The growing medium was composed by 80% w/w fine sand (< 0.5 mm) and 20% w/w sieved acid peat (< 2.5 mm). Part of the soil was spiked with a solution of sodium selenate (Na 2 SeO 4 ) or with sodium selenite (Na 2 SeO 3 ), to obtain a final soil concentration of 30 mg kg −1 Se DW. The remaining soil was moistened with deionized water and used as control. Spiked and control soils were stored in closed plastic bags for two weeks to allow equilibration. Selenium salts were purchased from Merck (Darmstadt, Germany).
Forty-eight rhizoboxes were prepared using squared 23 × 23 cm Petri dishes, each rhizobox hosted two replicate plants and in the central part of the rhizobox a polystyrene septum was placed to physically separate the rhizosphere of the two replicate plant individuals (Fig. 1).
Rhizoboxes were filled with control/selenate, control/selenite or control/control soils. Eight replicate plants per treatment were cultivated and, to take into consideration root chirality, in each rhizobox the Se-enriched soil was always placed in the central part so that for one plant was on the right and for the other on the left (Fig. 1). A rigid plastic sheet was used to separately fill each half of the same rhizobox, then removed to allow the contact of the two soil types.
Seedlings production and transfer. Selected Se-hyperaccumulators were S. pinnata (Pursh) (Brassicaceae) and A. bisulcatus (Hook.) A. Gray (Fabaceae) while non-accumulator plants B. juncea L. and M. sativa L. were selected as they belong to the same botanical family of the hyperaccumulators. Seeds of the Se-hyperaccumulators were purchased from Prairie Moon Nursery (Winona, Minnesota, USA) while those of non-accumulators were obtained from the Department of Agricultural and Food Sciences (University of Bologna, Bologna, Italy). Before germination, A. bisulcatus seeds were scarified using sandpaper. All seeds were hydrated with tap water for 4 h, then placed on moist paper for germination in the dark at 25 °C. The germination occurred in 5-7 days, then the seedlings were transferred in the rhizoboxes at cotyledon stage and watered with 1 ml of deionised water to assure establishment. Rhizoboxes were not watered anymore to avoid Se leaching or delocalization between the two halves and were covered with aluminium foil to avoid the light penetration in the soil. Then they were arranged with an angle of 45° to force roots growing on the Petri surface for observation. All plants were cultivated for 3 weeks in a growth chamber with 16 Sample collection and analysis. For each treatment, five g of soil from each rhizobox half were collected after the equilibration period (2 weeks) (initial soil) and at the end of the 3-weeks cultivation period (postcultivation soil). Soil samples were dried in a ventilated oven at 40 °C for 48 h and stored in plastic bags until analysis. Plant shoots and roots were separately collected. Roots from the two halves of the rhizobox could not be collected separately but were pulled together to achieve a total biomass suitable for further analysis. Plant parts were dried and stored similarly to soil. Aqua regia digestion was performed on soil samples to quantify total elements, following a modified method according to Hseu et al. 37 . 1.5 g DW of soil was weighted and placed in Teflon digestion tubes for close vessel digestion together with 6 ml of 37% v/v HCl, 2 ml of 69% v/v HNO 3 and 0.5 ml of 35% v/v H 2 O 2 . All the reagents at trace element analysis grade, were purchased from Merck (Darmstadt, Germany). Digestions were performed using a STARD D microwave system (Milestone, Sorisole, BG, Italy) with the following cycle: 2 min at 250 Watt, 2 min at 400 Watt, 1 min at 0 Watt and 3 min at 750 Watt. After digestion, all samples were brought to a volume of 20 ml with milliQ water.
For plant sample digestions, 0.1 g of dry weight (g DW) of roots/shoots were weighted and placed in Teflon digestion tubes with 69% v/v HNO 3 and 0.5 ml of 35% v/v H 2 O 2 following a modified method from Tüzen, (2003) 38 . Tubes were subsequently placed in the microwave and undergone the following digestion cycle: 2 min at 250 Watt, 2 min at 400 Watt, 1 min at 0 Watt and 2 min at 600 Watt. After digestion, samples were brought up to the volume of 10 ml. All samples were filtered with Whatman 42 ashless filter paper (Maidstone, UK).
The quantification of trace elements was carried out with a Spectro Arcos II ICP-OES (Ametek, Berwyn, Pennsylvania, US). The limits of quantification of the analysed elements were 0.067 mg kg −1 , 0.037 mg kg −1 , 0.042 mg kg −1 , 0.0054 mg kg −1 , 0.0065 mg kg −1 , 0.00031 mg kg −1 , 0.0017 mg kg −1 , 0.0113 mg kg −1 and 0.00038 mg kg −1 for Ca, K, Mg, Na, P, S, Fe, Se, and Zn, respectively. For quality control, BCR-143R sewage sludge amended soil (JRC-Joint Research Centre, Geel, Belgium) SRM 1570a spinach leaves (NIST, Maryland, US) were digested and analysed together with soil and plant samples. Recovery rates of certified elements were > 95%. Quality control solutions were also included during measurements to assure instrumental stability. Data were expressed as mg of element per kg sample dry weight (mg kgDW -1 ).
Selenium sequential extraction. Selenium sequential extraction was performed following a modified method from Chao et al. 22   www.nature.com/scientificreports/ F2, the residual soil from F1 was mixed with 25 ml of 0.1 M KH 2 PO 4 then shaken, centrifuged and filtered as before. To bring into solution F3, residual soil from F2 was mixed with 25 ml of 4 M HCl. Samples were incubated for 45 min at 95 °C then, centrifuged and filtered as above. F3 residual soil was oven-dried at 40 °C for 2 days and to quantify residual Se (F4), 1.5 g of each dried sample was weighted and analysed by ICP-OES as previously described.
Image processing. After three weeks of cultivation rhizoboxes were photographed with a Nikon D3200 camera (Chiyoda, Tokyo, Japan). All images were first modified using the software GIMP 2.10.32 (https:// www. gimp. org/), to homogenize the background soil, and then transformed into binary (only black and white pixels) using the Binary function of the software ImageJ 1.8.0 (https:// imagej. nih. gov/ ij/). Roots density (%) in each half of the rhizoboxes was calculated as the ratio of white pixel number / number of total pixel of each analysed half.
Data analysis. All variables were tested for homogeneity using Levene's test for homogeneity of variance and for normality using Shapiro-Wilk normality test from the package car (https:// cran.r-proje ct. org/ web/ packa ges/ car/ index. html). When data resulted parametric, analysis of variance (ANOVA) followed by Tukey HSD post-hoc test were used to evaluate differences among compared groups. When data resulted non-parametric, the analysis was performed using Research involving plants. The

Results
Root architecture in response to localized selenium. When grown in control/control and selenite/ control treatments, the Se-hyperaccumulator S. pinnata exhibited equal root density in both halves of the rhizobox (p = 0.53, p = 0.21) (Fig. 2a,b). However, in the presence of selenate, S. pinnata developed 76% of its roots towards the selenate-enriched half, and 24% in the control half (p < 0.05) (Fig. 2c). In contrast, B. juncea, the related non-accumulator of the same botanical family, did not show any preferential root distribution in either selenate (p = 0.78) or selenite (p = 0.98) treatments ( Fig. 2d-f).
A. bisulcatus Se-hyperaccumulator did not exhibit any sign of root foraging in selenite or selenate treatments, developing equal root density in both Se-enriched and control halves (p = 0.10, p = 0.61) (Fig. 3b,c). In control/ control rhizoboxes (Fig. 3a), A. bisulcatus roots were unevenly distributed in the two halves (77% and 13%, respectively, p < 0.05). Similarly to B. juncea, the non-accumulator M. sativa (of the same botanical family of A. bisulcatus), did not show any preferential root distribution in any treatment and was not sensitive to the presence of either selenite (p = 0.96) or selenate (p = 0.97) (Fig. 3d-f). In a few cases, both B. juncea and M. sativa showed signs of Se-avoidance, developing more roots in the control part than in the Se-enriched part, although these results were not statistically significant.
Soil elemental analysis. The total element concentration in soil is shown in Table 1. The average concentration of the analysed macronutrients before plant growth were 31,900 mg kgDW −1 Ca, 1300 mg kgDW −1 K, 2200 mg kgDW −1 Mg, 200 mg kgDW −1 Na, 160 mg kgDW −1 P and 550 mg kgDW −1 S. Overall, no significant differences were recorded after plant cultivation, except for a 4.8% decrease in the concentration of Ca in selenate/ control rhizobox with M. sativa (p < 0.05, Table 1). The initial concentrations of the Fe and Zn micronutrients was found to be on average 6700 and 25 mg kgDW -1 , respectively. Zinc decreased of the 23% after M. sativa cultivation only in control/control rhizoboxes (p < 0.05).
The average initial concentrations of Se were 22 and 23 mg kgDW −1 for selenite and selenate-enriched soils, respectively. A decrease in Se after A. bisulcatus and M. sativa cultivation was recorded both in selenite and selenate-enriched soils (average of 20 mg kgDW −1 , p < 0.05). An slight increase (+ 10%) in total Se was detected after the cultivation of S. pinnata but this was probably caused by an analytical error.
Selenium fractioning in soil. Selenium fractioning was evaluated in the soils before and after plant growth ( Fig. 4). Before plant cultivation, Se soluble fraction (F1) in the selenite and selenate-enriched soils accounted for the 35% and 37% respectively, the exchangeable Se (F2) was 8% and 7%, the oxide-bound Se (F3) accounted for 6% and 7%, while the residual fraction (F4) was the most abundant one (52% and 49%, in selenite and selenateenriched soil, respectively) (Fig. 4). Selenium was below the limit of detection (LoD) in control soils.
Overall, after plant cultivation the available fraction (F1) decreased of the 10-20% in all treatments. In particular in the hyperaccumulator S. pinnata, F1 passed from 36 to 15%, while F2 increased from 7.5% to 13.5% (average of selenate and selenite). F3 did not change after plant cultivation (p = 0.62), while F4 was significantly increased, passing from 50 to 65% (p < 0.05). In A. bisulcatus the depletion on F1 was less marked and accounted for the 11% for selenite and 3% in selenate treatments, while similar trends were detected other fractions. After Plant growth performances. Figure 5 represents the average shoot and root dry weight of the plant species measured at the end of the cultivation period in the three different treatments (control/control, selenate/ control, selenite/control). S. pinnata and B. juncea exhibited greater growth with an average harvestable shoot biomass among the three treatments of 0.24 and 0.36 gDW plant −1 respectively, compared to A. bisulcatus and M. sativa, with a shoot dry biomass of only 0.05 and 0.03 gDW plant −1 respectively, on average in the three treatments. A similar trend was observed for root biomass (Fig. 5) Selenium and sulphur accumulation. Selenium shoot concentration was found to be significantly higher in S. pinnata and A. bisulcatus compared to non-accumulators (p < 0.05) ( Table 2). Higher concentrations of this element were observed in selenate treatments (1950 and 2400 mg kgDW −1 ) compared to selenite ones (1320 and 530 mg kgDW −1 ,) in S. pinnata and A. bisulcatus shoots respectively. A similar trend was observed for shoot and root Se concentration of non-accumulator species (Table 2). Sulphur was also measured since its accumulation is closely linked to that of Se and differs in hyperaccumulator and non-accumulator plants. Overall,

Discussion
Only S. pinnata plants cultivated in the selenate/control rhizoboxes showed significant evidence of root foraging, producing most of the roots (75%) in the selenate-enriched soil (Fig. 2). This result was in line with previous results 36 which reported root foraging in S. pinnata when cultivated for two months in pots. Conversely, A. bisulcatus (Fig. 3), did not show any preferential distribution of roots towards the Se-enriched part (either selenate or selenite) of the rhizobox as also observed by Goodson et al. 36 , who reported a main vertical development of roots at the expenses of lateral branching. Other Se-hyperaccumulators have been reported to forage Se, including Symphyotrichum ericoides (L.) G.L.Nesom which showed a preferential root development towards Se, in terms of biomass, length and width 39 . Recently, also Neptunia amplexicaulis, a Se-hyperaccumulator native of Queensland (Australia), was reported to forage Se when this element was in the soil in soluble form 35 . The present study also reported that some individuals of non-accumulators B. juncea and M. sativa showed signs of avoidance, having a lateral root proliferation shifted towards the control soil (not statistically significant). This type of behaviour in B. juncea was never reported in previous studies which analysed root architecture of this species under similar conditions 36 . On the other hand, Medicago sativa, the second non-accumulator plant here used, was never tested before for Se foraging.
Plant growth did not have a significant impact on soil macro-and micro-nutrients because of the short period of plant cultivation (Table 1). Nevertheless, the plant growth impacted on Se content and fractioning. The Se concentration in soil before cultivation resulted considerably lower than expected 22 and 23 mg kgDW -1 instead of 30 mg kgDW -1 for both selenite and selenate initially provided during soil preparation. This could have been caused by Se volatilization probably occurring during the equilibration period (e.g. methylation by soil microorganisms) 1,20 or during soil processing prior to analysis (e.g. drying and mineralization).
Selenium distribution in the different fractions differed between selenite and selenate enriched soil (Fig. 4). After plant cultivation in the selenite soils, the residual fraction (F4) accounted for the 69% of the total, compared to the 48% of selenate soils. Soluble and exchangeable Se forms (F1-F2) were in fact preferentially removed by plant roots, triggering Se release from less mobile fractions (i.e. F4) 18 . In line with this, soluble Se (F1) was higher in soils spiked with selenate (29%, on average) than in those with selenite (14%, on average). Selenate is in fact known to be less toxic and more absorbed by plant roots with respect to selenite 10,18 . Since soluble Se represents the most available form for plants 18,40 , fraction F1 significantly decreased (about − 25%) after S. pinnata cultivation, especially in the soils spiked with selenate. Plants can occasionally extract Se from F3-F4 fractions, for examples when roots come in direct contact with this pool 18 . However due to restricted root development this effect was not observed in the present experiment. Overall, Se distribution in residual fraction (F4) and oxidebound fraction (F3) resulted in line with the values found soil amended with 0.5 mg kgDW −1 selenite after rice (Oryza sativa L.) cultivation 41 (60-80% of Se in F4 and 4-10% in the F3). As further confirmation, plant biomasses were larger in selenate/control rhizoboxes compared to selenite ones for all the tested species including non-accumulators (Fig. 5). Similar results were obtained for lettuce (Lactuca sativa L.) and cucumber (Cucumis sativus L.) whose biomass decreased of 60% more in plants exposed to selenite compared to those subjected to the same selenate concentration 42,43 .
As expected hyperaccumulators, especially S. pinnata, produced a larger shoot biomass (+23%) in presence of Se, whereas non-accumulator plants showed the opposite trend (Fig. 5). Previous studies confirmed that Se represents a beneficial element for hyperaccumulators stimulating their growth 10,44 .
Selenium accumulation in S. pinnata and A. bisulcatus was higher compared to the non-accumulators (Table 2) and both species accumulated more Se when cultivated on selenate-enriched soils compared to the selenite-enriched ones. The Se concentrations reached in shoots of these plants (1950 and 2400 mg kgDW −1 , respectively) after only 3 weeks of cultivation on 30 mg/kg DW Se spiked soil, resulted comparable to those detected in the same plants grown under natural conditions 45,46 . www.nature.com/scientificreports/ Interestingly, when cultivated on selenate-enriched soil, also B. juncea accumulated notable quantities of Se (476 mg kg DW −1 in shoots) ( Table 2). This result was in line with previous studies carried out on the same species, with an accumulation of about 500 mg kgDW −1 Se when grown for 4 weeks on soil treated with 20 µM selenate 47 . Due to its ability to store significant quantities of Se in the shoot biomass, B. juncea has been sometimes reported as a secondary Se accumulator 27 . Lastly, M. sativa resulted the least efficient species in Se accumulation with 297 mg kgDW −1 Se in shoots after growth on soil spiked with selenate (Table 2). Similarly, a previous study demonstrated that this species grown for longer period (6 months) on soil spiked with 20 mg kgDW −1 selenate never overcame 600 mg kgDW −1 Se in its shoots 48 . The present results showed that in selenate treatment the Se/S ratio was 30-40% higher in hyperaccumulator than in non-accumulator plant species. It has in fact been demonstrated that Se-hyperaccumulators, although using the same strategy than non-accumulators in taking up Se, seem to be able to distinguish between Se and S 27 . This capacity results in much higher Se/S ratio in their tissues compared to non-accumulator species 49 .

Conclusions
The present study provides new insights on the ability of the Se-hyperaccumulators S. pinnata and A. bisulcatus to and the non-accumulators B. juncea and M. sativa to forage for Se, and may have important implications for the future development of strategies aimed at managing Se in contaminated soils or for crop biofortification. Our results suggest that Se has a growth promoting effect on the hyperaccumulator S. pinnata, especially if provided in the form of selenate. S. pinnata proved to actively forage for selenate under the tested conditions, whereas this phenomenon was not observed when the plant was cultivated with selenite. A. bisulcatus, did not forage for Se as its root development was not affected by the presence of Se in both forms. At the tested concentrations, nonaccumulators did not show any significant sign of Se toxicity in response to Se addition in either form. However, B. juncea and M. sativa showed occasionally signs of Se avoidance when cultivated in rhizoboxes with selenate, with roots developing preferentially in the control half.
Our results showed that selenate is more easily detected and preferentially taken up by plant roots compared to selenite and that the soluble and exchangeable fractions (F1, F2) are readily utilized and removed by plants.
To further extend the actual understanding of the factors influencing Se foraging and accumulation in plant species, future studies should focus on investigating the role of both biotic and abiotic factors in the uptake and accumulation of Se, such as exploring the plant-microbe interactions and the impact of pH, soil type, and irrigation practices.

Data availability
All data generated during the current study and additional pictures are available from the corresponding author upon reasonable request.